Determination of the microbial community of traditional Mongolian cheese by using culture‐dependent and independent methods

Abstract Mongolian cheese is not only a requisite source of food for the nomadic Mongolian but also follows a unique Mongolian dairy artisanal method of production, possessing high nutritional value and long shelf‐life. In this study, the ancient technique for the production of Mongolian cheese was investigated. The nutritional value of Mongolian cheese was characterized by its high‐protein content (30.13 ± 2.99%) and low‐fat content (9.66 ± 3.36%). Lactobacillus, Lactococcus, and Dipodascus were the predominant bacterial and fungal genera, and Lactobacillus helveticus, Lactococcus piscium, and Dipodascus geotrichum were the predominant species in the Mongolian cheese. The microbiota of products from different cheese factories varies significantly. The high‐temperature (85°C–90°C) kneading of coagulated curds could eliminate most of the thermosensitive microorganisms for extending the shelf‐life of cheese. The indigenous spore‐forming microbes, which included yeasts, belonging to Pichia and Candida genera, and molds, belonging to Mucor and Penicillium genera, which originated from the surroundings during the process of cooling, drying, demolding, and vacuum packaging could survive and cause the package to swell and the cheese to grow mold. The investigation of production technology, nutrition, microbiota, and viable microbes related to shelf‐life contributes to the protection of traditional technologies, extraction of highlights (nutritional profiles and curd scalding) for merchandise marketing, and standardization of Mongolian cheese production, including culture starters and aseptic technique.

traditional craft (Figure 1). Nevertheless, the ancient technique of Mongolian cheese production has rarely been investigated.
Cheese making begins with the natural fermentation of raw milk filtered using multilayered gauze (Figure 1a). The raw milk comes from cows, sheep, and camels. The natural fermentation continues spontaneously at ambient temperature for 1-2 days (Figure 1b). The end of natural fermentation depends on the sensory experience of the maker such as the sour taste of semisolid gels (curds), the appearance of upper sour cream, and the firmness of curd. After removing the upper sour cream (Zuohe), the curd is transferred from the vat to a pot, which is heated using a low flame for separating the coagulated curd followed by draining the whey with the help of scoop ( Figure 1c). The coagulated curd is then heated using a high flame and kneaded into a high-fluid state followed by the removal of whey (Figure 1d). The fluid cheese is molded into a wooden box, which is then cooled and dried (Figure 1e). The abundant field in- vestigation for the production of cheese showed that the temperature of natural fermentation (18-20°C) was lower than the average room temperature, where the fermentation vat was kept in a cool closet in dark; the natural fermentation may reach 75°T (acidity) and pH 4.0 at the end. In addition, the curd is heated to 40-60°C for removing the whey, while the coagulated curd is heated to 85-90°C and kneaded continuously for producing a smooth and fluid cheese.
The ambient temperature ensures the continuation of natural fermentation till the end and endows a distinct flavor into Mongolian cheese by increasing flavor-producing substances. The duration of fermentation depends on the microbiota of raw milk, sanitary conditions of vat and atmosphere, and ambient temperature. This ar- tisanal production process of Mongolian cheese, including acidity and temperature of fermentation, draining of whey, and kneading of coagulated curd contributes to the standardized production of Mongolian cheese.
As the studies on Mongolian cheese might increase and win universal praise, the fundamental investigations, including artisanal production method, nutrition, and microbiota are needed to be carried out. In the present study, the traditional production of Mongolian cheese was investigated in field practices and its quantification indicators and nutritional contents were measured using a large number of samples followed by the identification of microbial composition, including bacteria and fungi. Last, the viable microbes in the Mongolian cheese were quantified, isolated, and identified to investigate the underlying reasons, affecting its shelf life.

| 16 S rRNA and ITS gene sequencing, bioinformatics, and statistical analysis
The total microbial DNA was extracted and purified using E.Z.N.A stool DNA kit (Omega Bio-tek, Norcross, US). The V3-V4 hypervariable region of 16 S rRNA gene was amplified with 341F 5′-CCTACGGGNGGCWGCAG-3′ and 806R 5′-GGACTACHVGGGT ATCTAAT-3′ primers. The ITS gene sequence was amplified with ITS3-KYO2F 5′-GATGAAGAACGYAGYRAA-3′ and ITS4R 5′-TCCT CCGCTTATTGATATGC-3′. The PCR reaction mixture consisted of the following: 5 μl of 10 X KOD buffer, 1 μl of KOD polymerase, 5 μl of 2.5 mM dNTPs, 1.5 μl of each primer (5 μM), and 100 ng of microbial DNA. The thermal program of PCR was as follows: initial denaturation at 95°C for 2 min; followed by 27 cycles of denaturation at 98°C for 10 s, annealing at 62°C for 30 s, and extension at 68°C for 30 s; and final extension at 68 °C for 10 min. The amplicons were quantified and subjected to paired-end sequencing (2 × 250) using the Illumina MiSeq platform (Illumina, San Diego, CA). The high-quality sequencing reads were acquired by removing the reads with more than 10% of unknown nucleotides and those with less than 80% of bases with quality-value (Q-value) >20. The final effective reads were obtained by trimming the chimeric tags and were clustered into the operational taxonomic units (OTUs) with ≥97% similarity using the UPARSE pipeline (Edgar, 2013). The OTUs were classified into organisms with the Naive Bayesian Model using RDP classifier (Wang et al., 2007) based on SILVA database for 16 S rRNA gene sequencing (Pruesse et al., 2007) and UNITE database for ITS gene sequencing (Koljalg et al., 2005).

F I G U R E 2 Physicochemical composition and viable microbial dynamics
during the production of Mongolian cheese. Sampling of Mongolian cheese in the five administrative divisions of inner Mongolia and its geographical position in China is highlighted (a). Contents of moisture, protein, and fat were measured as per the national food safety standards in China (b). Dynamics of LAB, yeast, and mold counts were demonstrated via histogram during the production of Mongolian cheese at three time points (Figure 1a,b,d) from three cheese factories (c)

| Nutritional and microbiological investigation of Mongolian cheese
There are approximately 18 types of cheese varieties based on the classification scheme of production methods, including curding, cutting the coagulum, stirring, draining, heating, pressing, salting, and ripening (Walter & Hargrove, 1972). In general, Mongolian cheese is classified as an acidic curd cheese for the natural fermentation of raw milk to 75°T (acidity) and pH 4.0 ( Figure 1). The acidcoagulated Mongolian cheese is characterized by high moisture (53.65 ± 3.46%) and protein contents (30.13 ± 2.99%) and low-fat content (9.66 ± 3.36%) for cream floating during natural fermentation ( Figure 1b). The superficial floating cream in coagulum is named Zuohe in Mongolian. In order to fully understand the conventional nutritional potential of Mongolian cheese, a total of 32 samples were collected from the Mongolian nomads in the major cheeseproducing areas including Xilin Gol, Chifeng, Tongliao, Hinggan, and Hulun Buir (Figure 2a). The moisture content ranged from 47.77% to 58.59%, and the protein and fat contents ranged from 25.07% to 34.96% and from 2.36% to 14.98%, respectively ( Figure 2b). The Mongolian nomads heat coagulum and stir it to a high-fluid state based on their experience and preference and usually consume or sell it soon after production. The intensity of removing whey, stirring, and drying was slightly different from the artisanal production methods by Mongolian nomads and determined the moisture content within limits. The changes in acidity during natural fermentation could influence the degree of protein coagulation and cream floating, thereby affecting the corresponding contents.
The changes in microbial abundance in the manufacturing process of Mongolian cheese were analyzed by sampling at three time points (Figure 1a, b) from three cheese factories. The total LAB count ranged from 1.17 × 10 6 , 9.65 × 10 5 , and 1.29 × 10 6 cfu/g in the raw milk (Figure 1a) to 1.31 × 10 12 , 3.41 × 10 12 , and 1.28 × 10 12 cfu/g at the end of fermentation, respectively (Figure 1b), while the LAB count was 0 cfu/g in the Mongolian cheese that had just been produced ( Figure 1). The yeast count ranged from 6.45 × 10 4 , 1.79 × 10 4 , and 7.3 × 10 3 cfu/g in the raw milk to 5.9 × 10 5 , 1.5 × 10 4 , and 1.1 × 10 4 cfu/g at the end of fermentation, respectively, while the yeast count was 0 cfu/g in Mongolian cheese. Similarly, the mold count ranged from 100, 50, and 2.25 × 10 3 cfu/g in the raw milk to

| Abundance and composition of microbiota in Mongolian cheese
Although the Mongolian cheese, which had just been produced, did not have viable LAB, yeast, and mold, these microorganisms play a vital role in the formation of coagulated curd and flavor of cheese.
The composition of the microbiota, including bacteria and fungi, which existed during the production of cheese were investigated using 16 S rRNA and ITS genes sequencing technology. The Shannon curves, but not the rarefaction curves, reached saturation in the sequencing of 16 S rRNA and ITS genes (Table S1 and Figure S2). This indicated that the sequencing depth was sufficient to represent the whole bacterial and fungal communities. There were significant differences in the diversity of bacterial and fungal communities among Mongolian cheese from the three artisanal factories (p < 0.001), while the microbial diversity did not alter during the three different periods of production by the same manufacturer (p > 0.05). The different sources of raw milk and production atmospheres showed that the bacterial and fungal communities were involved in natural fermentation, thereby ultimately affecting the coagulation.

| Quantification, isolation, and identification of viable microbes from Mongolian cheese
A great diversity of bacterial and fungal species were discovered in the Mongolian cheese using amplicon sequencing. The microbiota in Mongolian cheese contained all the microorganisms residing in raw milk as well as those surrounding the environment of production, which were eliminated using high temperatures (85-90°C) (Figure 1d and Figure 2c). Nevertheless, cheese could easily get mildewed and its wrapping was liable to swell up ( Figure 5).
It was speculated that the viable microbes, which affect the shelf life of cheese, might originate from the surroundings during the process of cooling, drying, demolding, and wrapping. In order to verify this hypothesis, the artisanal cheese from the three different factories was divided into three parts (the outer part; the middle part; the inner part) from the outside to the inside ( Figure 6). The TBC, LAB, yeast, and mold counts were determined using culturedependent methods in the samples from the three parts. Figure 7a shows that the TBC and LAB counts in the outer part of cheese from Fenghua were significantly more as compared to those in the middle and inner parts (p < 0.05), while the yeast and mold did not exist in the middle and inner parts. The species diversity and abundance of TBC did not change from the outer to the inner layer of cheese (Figure 8a), while the diversity of LAB decreased from the outer to the middle layer of cheese ( Figure 8b). As shown in Figure 7b, the TBC and LAB count in the outer part of cheese from F I G U R E 6 Cheese was divided into three parts (the outer part; the middle part; the inner part) from the outside to the inside F I G U R E 7 The TBC, LAB, yeast, and mold counts were determined using culture-dependent methods in the samples from the outer part, the middle part, and the inner part of cheese (a: Fenghua; b: Suainiu; c: Muxiangyuan) Suainiu was significantly more as compared to those in the middle part (p < 0.05), while yeast and mold did not exist in the middle and inner parts. The diversity of TBA and LAB decreased from the outer to the middle layer of cheese (Figure 8c,d). As shown in Figure 7, the TBC and yeast counts in the outer part of cheese from Muxiangyuan were significantly more than those in the middle and inner parts (p < 0.05), while LAB and mold did not exist in the middle and inner parts. The diversity of TBC and yeast decreased from the outer to the middle layer of cheese (Figure 8e,f). In conclusion, the indigenous bacteria and fungi, which existed in the environment of production, including cooling, drying, demolding, and wrapping, colonized the surface of cheese, causing the swelling of packages and the formation of mold. The yeast species, belonging to Pichia and Candida, can produce carbon dioxide, and the molds, belonging to Mucor and Penicillium, grow on the surface of cheese.
Some spore-forming molds also overcome high temperatures (85-90°C) and survive, affecting the shelf life of cheese.

| CON CLUS IONS
Mongolian cheese has been a cultural icon of food since the ancient days. In this study, the scientific implications of the ancient technique for the production of Mongolian cheese were explored. The contents of protein and fat were 30.13 ± 2.99% and 9.66 ± 3.36%, respectively.
The high-temperature (85-90°C) kneading of coagulated curds could eliminate most of the thermosensitive microorganisms in cheese.
The predominant bacterial and fungal genera included Lactobacillus, F I G U R E 8 The species diversity and abundance of TBC, LAB and yeast were determined using ITS sequencing method in the samples from the outer part, the middle part and the inner part of cheese (a and b: fenghua; c and d: Suainiu; e and f: Muxiangyuan) Lactococcus, and Dipodascus, and Lactobacillus helveticus, Lactococcus piscium, and Dipodascus geotrichum were predominant species in the Mongolian cheese. The vacuum-sealed plastic packs of Mongolian cheese are liable to mildew and swell. Using the quantification, isolation, and identification of microbes in the different spatial positions of cheese, it was found that the indigenous spore-forming microbes, which survived at high temperature (85-90°C) and included yeasts, belonging to Pichia and Candida, and molds, belonging to Mucor and Penicillium, originated from the surroundings during the process of cooling, drying, demolding, and wrapping and might cause the vacuum packages to swell and make the cheese to easily get mildew.
According to the experimental results, it was assumed that sterilizing the surroundings of cooling, drying, demolding, and packaging, use of antibacterial packaging materials, and heat sterilization after vacuum packaging might eliminate the microbes on the surface of cheese to some extent, thus increasing its shelf-life.

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.